An amplification unit in human melanoma cells showing partial homology with sequences of human papillomavirus type 9 and with nuclear antigen 1 of the Epstein-Barr virus.

By partial homology with the DNA of human papillomavirus type 9 a cellular amplification unit was detected which is amplified in melanoma cells but not in Epstein-Barr virus-transformed B cells of two melanoma patients. A 2.4-kilobase EcoRI fragment of this amplification unit was cloned and designated mel/HPV9. At the chromosomal level we detected mel/HPV9 in homogeneously staining regions or in abnormally banded regions containing different marker chromosomes of both melanoma cell lines. DNA sequence analysis of a part of mel/HPV 9 revealed homology with the third internal repeat array of Epstein-Barr virus nuclear antigen 1.

Transcriptional and translational downregulation of H-REV107, a class II tumour suppressor gene located on human chromosome 11q11-12.

The H-rev107 tumour suppressor was isolated as a gene specifically expressed in rat fibroblasts resistant toward malignant transformation by the activated HRAS gene (Sers et al., 1997; Hajnal et al., 1994). Here we describe the human homologue of the rat H-rev107 gene. The predicted rat and human proteins are highly conserved exhibiting an overall amino acid identity of 83%. The H-REV107-1 gene is ubiquitously expressed with the exception of haematopoetic cells and tissues. In contrast, H-REV107-1 mRNA was found only in eight of 27 cell lines derived from mammary carcinoma, lung carcinoma, gastric carcinoma, kidney carcinoma, melanoma, neuroblastoma and other tumours. The H-REV107-1 protein was not detectable in any of these tumour cells. Loss of H-REV107-1 expression was not restricted to cultured human tumour cell lines, but also found in primary squamous cell carcinomas. Gross structural aberrations of the H-REV107-1 gene were absent in tumorigenic cell lines. Thus, the block to H-REV107-1 expression is achieved both at the level of transcription and translation. By fluorescence in situ hybridisation the human H-REV107-1 gene was localised to chromosome 11q11-12.

Predetermined chromosomal deletion encompassing the Nf-1 gene.

Complex chromosomal rearrangements (deletions, inversions, translocations) are a hallmark of human tumour cells. Yet, the generation of animal models for gross chromosomal abnormalities still presents a formidable challenge. Here, we describe a versatile procedure for chromosomal engineering that was used to generate an ES cell line with a megabase deletion encompassing the tumour suppressor gene neurofibromatosis-1 (Nf-1) on mouse chromosome 11, which is often deleted in tumours of neural crest origin. Homologous recombination into sites flanking Nf-1 was used to introduce artificial sequences (triple-helix, loxP, vector backbone) that can be employed for in vitro recovery of intervening sequences or the generation of in vivo deletions. This strategy may be developed into a scheme by which large chromosomal regions with precisely defined end points may be excised from mammalian cells and reintroduced after suitable in vitro modification.

Primary structure, chromosomal mapping, expression and transcriptional activity of murine hepatocyte nuclear factor 4gamma.

We demonstrate the presence of a new member of the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) subfamily in mouse which is genetically distinct from the previously characterized mouse HNF4alpha gene. The new member of the HNF4 subfamily shows highest amino acid identity, similar tissue distribution and syntenous chromosomal localization to the recently described human HNF4gamma (NR2A2), we therefore classify it as mouse HNF4gamma (mHNF4gamma). A combination of RT-PCR and immunohistochemical analysis showed expression of mHNF4gamma mRNA and protein in the endocrine pancreas, testes, kidney and gut. By co-transfection experiments, we show that mHNF4gamma is able to activate transcription, acting through binding sites that have been previously characterized as HNF4alpha binding sites. The presence of HNFgamma in human and mouse implies that a complex transcriptional network exists in higher vertebrates involving a number of HNF4 members with overlapping yet distinct function and tissue distribution.

Pbx4, a new Pbx family member on mouse chromosome 8, is expressed during spermatogenesis.

Members of the Pbx family are involved in a diverse range of developmental processes including axial patterning and organogenesis. Pbx functions are in part mediated by the interaction of Pbx proteins with members of the Hox and Meis/Prep families. We have identified a fourth mammalian Pbx family member. Pbx4 in the mouse and PBX4 in humans are located on chromosome 8 and chromosome 19, respectively. Pbx4 expression is confined to the testis, especially to spermatocytes in the pachytene stage of the first meiotic prophase.

The mouse filensin gene: structure and evolutionary relation to other intermediate filament genes.

Filensin and phakinin are two lens-specific members of the intermediate filament (IF) superfamily of proteins. They coassemble to form a beaded submembraneous filamentous network, the beaded filaments (BFs). The low sequence homology and differences in assembly compared to other IF proteins do not allow their classification in any of the five IF subgroups. The organization of the phakinin gene exon/intron boundaries provides evidence that this partner may be sharing a common origin with type I cytokeratin genes. Here we report the molecular cloning, sequence and characterization of the mouse filensin gene. The filensin gene consists of 8 exons and 7 introns, with 6 introns interrupting its rod domain in a highly conserved manner characteristic of type III IF genes, like vimentin, desmin, or peripherin. Of the two tail domain exons the one adjacent to the rod domain, compares to exon 7 of the non-neuronal cytoplasmic IF gene of helix aspersa and to the lamin region bridging the end of the rod domain to the nuclear localization signal. Altogether, these observations indicate that the lens beaded filaments form an independent class of IF.

Murine epidermal lipoxygenase (Aloxe) encodes a 12-lipoxygenase isoform.

Using a combination of conventional screening procedures and polymerase chain reaction cloning, we have isolated a cDNA encoding an epidermis-type 12-lipoxygenase (e12-lipoxygenase) from mouse epidermis. The open reading frame corresponds to a protein of 662 amino acids and was found to be 99.8% identical to the ORF of an epidermal lipoxygenase gene Aloxe, described recently [Van Dijk et al. (1995) Biochim. Biophys. Acta 1259, 4-8]. When expressed in human embryonic kidney cells the recombinant protein could be shown to synthesize 12(S)-HETE from arachidonic acid. By fluorescence in situ hybridization the e12-lipoxygenase gene was localized to chromosome band 11 B1-B3.

Human homeodomain-interacting protein kinase-2 (HIPK2) is a member of the DYRK family of protein kinases and maps to chromosome 7q32-q34.

Here we identified the human serine/threonine kinase HIPK2 as a novel member of the DYRK kinase subfamily. Alignment of several DYRK family proteins including the kinases minibrain, MJAK, PKY, the Dictyostelium kinase YakA and Saccharomyces YAK1 allowed the identification of several evolutionary conserved DYRK consensus motifs within the kinase domain. A lysine residue conserved between all DYRK kinase family members was found to be essential for the kinase function of HIPK2. Human HIPK2 was mapped to chromosome 7q32-q34 and murine HIPK2 to chromosome 6B, the homologue to human chromosome 7.

Genomic organization of mouse gene zfp162.

We report the cloning and characterization of the alternatively spliced mouse gene zfp162, formerly termed mzfm, the homolog of the human ZFM1 gene encoding the splicing factor SF1 and a putative signal transduction and activation of RNA (STAR) protein. The zfp162 gene is about 14 kb long and consists of 14 exons and 13 introns. Comparison of zfp162 with the genomic sequences of ZFM1/SF1 revealed that the exon-intron structure and exon sequences are well conserved between the genes, whereas the introns differ in length and sequence composition. Using fluorescent in situ hybridization, the zfp162 gene was assigned to chromosome 19, region B. Screening of a genomic library integrated in lambda DASH II resulted in the identification of the 5'-flanking region of zfp162. Sequence analysis of this region showed that zfp162 is a TATA-less gene containing an initiator control element and two CCAAT boxes. The promoter exhibits the following motifs: AP-2, CRE, Ets, GRE, HNF5, MRE, SP-1, TRE, TCF1, and PU.1. The core promoter, from position -331 to -157, contains the motifs CRE, SP-1, MRE, and AP-2, as determined in transfected CHO-K1 cells and IC-21 cells by reporter gene assay using a secreted form of human placental alkaline phosphatase. The occurrence of PU.1/GRE supports the view that the zfp162 gene encodes a protein involved not only in nuclear RNA metabolism, as the human ZFM1/SF1, but also in as yet unknown macrophage-inherent functions.

An alternative approach for induction of chromosomal banding and preservation of chromosomal morphology for virus DNA mapping by in situ hybridization.

I report on the use of acetic/saline/Giemsa technique for the induction of chromosome banding and an additional fixation (prior hybridization) to preserve the chromosome morphology as regards in situ hybridization under stringent conditions. This approach results in high quality banding resolution for grain localization of integrated sites of virus DNA sequences on both metaphase and prometaphase chromosomes.

Induction of chromosomal aberrations in human cells by a temperature-sensitive mutant of herpes simplex virus type 2 and its revertants.

The induction of chromosomal aberrations by a temperature-sensitive (ts) mutant of herpes simplex virus type 2 (HSV-2) strain Hg52 (ts 13), its revertants 4-8 and 5-8 and by etalon strains HSV-1 17 syn+ and HSV-2 Hg52 was studied in human fibroblast and lymphocyte cultures. The effect on chromosomes of the revertants was tested at permissive (31 degrees C) and non-permissive (38 degrees C) temperatures. At 38 degrees C the revertants could not induce DNase activity. The present results contribute to the possible role of a herpes-coded nuclease in induction of chromosomal aberrations.

Effects of herpes simplex virus strains on human fibroblast and lymphocyte chromosomes and the localization of chromosomal aberrations.

The effects on human chromosomes of types 1, 2 and of intermediate serotype of herpes simplex virus (HSV) was compared in fibroblast and lymphocyte cultures. The karyological changes due to HSV were shown to depend on the serotype used as well as on the kind of cells examined (agent-specificity and cell-reaction specificity). Differences were noted among the strains in relation to the degree and character of the aberrations induced. Conventional Giemsa staining and the trypsin G-banding techniques were used to localize aberrations in the length of human fibroblast and lymphocyte chromosomes after HSV infection. A non-random damage of chromosomes 1 and 3 displaying the same pattern in either cell type was established. The distribution of chromosomal abnormalities was independent of the chromosome length. The topographic banding analysis of lesions induced by strains of HSV-1, HSV-2 and intermediate serotypes showed that the most frequent aberrations were localized in bands p32, p34, q21 and q32 of chromosome 1 and in the band q21 of chromosome 3. The localization of the most frequently occurring aberrations in the chromosomes belonging to other groups was also determined.

Detection of human papillomavirus DNA in gynaecological swabs by filter in situ hybridization.

Gynaecological smears from the endo- and ectocervix of women with and without cytological and colposcopic abnormalities of the epithelium were investigated for human papillomavirus (HPV) types 6, 11, 16, and 18 by filter in situ hybridization (FISH). The data were compared with cytological, colposcopic, and histological findings. Of the 266 gynaecological smears, HPV DNA was detected in 84 (32%); of 101 cytologically and colposcopically HPV negative cases, HPV DNA was found in 10%. Of 56 women, cytologically and colposcopically positive for HPV infection, HPV DNA was detected in 68%. The sensitivity of the method was controlled by comparing the results of FISH with those of Southern-blot analysis of five cervical tumour biopsies. The data presented demonstrate the necessity of FISH for identification of the HPV type that might be of prognostic value in cervical pathology. Cytological and colposcopic positivity is a reliable sign in about 70% of the cases where HPV infection was proved by FISH.

[The demonstration of human papillomavirus DNA in genital lesions with and without colposcopic atypism]. / Dokazvane na choveshka papilomavirusna DNK pri genitalni lezii sus i bez kolposkopski atipizum.

34 cellular suspensions from the vaginal part of women were examined for presence of HPV DNA of the types: 6, 11, 16 and 18--from a lesion and a healthy area--by filter in situ hybridization. The women were examined by colposcopy because of cervical lesions established macroscopically as well as for other various complaints. Cells were also investigated from women after undergone ablation treatment: diathermy coagulation, conization and hysterectomy on account of CIN. The data were compared with cytological, colposcopic and histological findings. The established presence of HPV DNA of high risk type 16 and 18 in some of the patients after operative treatment required continuous control of these women as well as examination of lower genital tract because of the discovered multicentricity of HPV infection.

[The demonstration of HPV DNA in cervical intraepithelial neoplasms and in invasive carcinomas of the cervix uteri by DNA-DNA in situ hybridization]. / Dokazvane na DNK na HPV v tservikalni intraepitelni neoplazii i v invazivni kartsinomi na matochnata shiika chrez DNK-DNK in situ khibridizatsiia.

Authors were applied a method of DNA-DNA in situ hybridization for demonstration of HPV 6, 11, 16 and 18 in cervical neoplasia and in invasion carcinoma of uterine neck. At 22 probes from CYV III and invasion carcinoma the results from hybridization with virus DNA were shown the presence of HPV-DNA at 15 patients. Some phases of method were optimized. Sensitiveness of method was controlled and compared with the results from the same probes, underlied to investigation with Southern-blot hybridization.

Comprehensive genomic analysis of desmoplastic medulloblastomas: identification of novel amplified genes and separate evaluation of the different histological components.

Desmoplastic medulloblastoma (DMB) is a malignant cerebellar tumour composed of two distinct tissue components, pale islands and desmoplastic areas. Previous studies revealed mutations in genes encoding members of the sonic hedgehog pathway, including PTCH, SMOH and SUFUH in DMBs. However, little is known about other genomic aberrations. We performed comparative genomic hybridization (CGH) analysis of 22 sporadic DMBs and identified chromosomal imbalances in 20 tumours (91%; mean, 4.9 imbalances/tumour). Recurrent chromosomal gains were found on chromosomes 3, 9 (six tumours each), 20, 22 (five tumours each), 2, 6, 7, 17 (four tumours each) and 1 (three tumours). Recurrent losses involved chromosomes X (eight tumours), Y (six of eleven tumours from male patients), 9, 12 (four tumours each), as well as 10, 13 and 17 (three tumours each). Four tumours demonstrated high-level amplifications involving sequences from 1p22, 5p15, 9p, 12p13, 13q33-q34 and 17q22-q24, respectively. Further analysis of the 9p and 17q22-q24 amplicons by array-based CGH (matrix-CGH) and candidate gene analyses revealed amplification of JMJD2C at 9p24 in one DMB and amplification of RPS6KB1, APPBP2, PPM1D and BCAS3 from 17q23 in three DMBs. Among the 17q23 genes, RPS6KB1 showed markedly elevated transcript levels as compared to normal cerebellum in five of six DMBs and four of five classic medulloblastomas investigated. Finally, CGH analysis of microdissected pale islands and desmoplastic areas showed common chromosomal imbalances in five of six informative tumours. In summary, we have identified several novel genetic alterations in DMBs and provide genetic evidence for a monoclonal origin of their different tissue components.

[The CO2 laser is an effective method for treating condylomata acuminata]. / CO2-lazer efektiven metod za lechenie na condilomata acuminata.

Twenty-six patients presenting condylomata acuminata are operated with CO2 laser (two in outpatient conditions), and 22 of them (84.61 per cent) estimate the pain as mild. Recurrence of the disease is registered in one case (3.85 per cent), but most likely it is a matter of minor vegetations, missed during the operation. In extensive condylomatous vegetations, CO2 laser is applied in conjunction with electrocautery in a fashion to obviate the side effects relating to electroresection. In five patients the laser radiation toxic effect on HPV is demonstrated for the first time by the filtered in situ hybridization method. It is worth noting that the therapeutic approach suggested combines radicality and efficiency of treatment, with the patients running an uneventful and painless postoperative course.

Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization.

Metaphase chromosomes of three cervical cancer cell lines (HeLa, CasKi, SiHa) were subjected to in situ hybridizations with the DNA of human papillomaviruses (HPV) types 16 and 18, respectively. Previous studies have demonstrated multiple copies of HPV 18 DNA in HeLa and of HPV 16 DNA in CasKi cells, but only 1-2 HPV 16 copies in cells of the SiHa line. The viral DNA persists in an integrated state (Schwarz et al 1985). Analysis of the integration sites revealed at least 11 chromosomal sites of HPV 16 integration in CasKi cells. SiHa cells contain integrated HPV 16 DNA in the region q21-q31 of chromosome No. 13. In HeLa cells integration of HPV 18 occurred in chromosome No. 8, band q24. Thus, no evidence was obtained for the existence of preferential chromosomal regions for HPV integration. The data indirectly support a trans-acting function of HPV-mediated cell transformation.